WO2018223760A1

WO2018223760A1 - Electrochemical biosensor electrode, sensor and preparation method therefor

Info

Publication number: WO2018223760A1
Application number: PCT/CN2018/082237
Authority: WO
Inventors: 冯雪; 鲁思渊; 陈毅豪; 方旭飞; 苏红宏
Original assignee: 清华大学
Priority date: 2017-06-09
Filing date: 2018-04-09
Publication date: 2018-12-13
Also published as: CN107422012B; CN107422012A

Abstract

An electrochemical biosensor electrode, a sensor and a preparation method therefor. The sensor electrode comprises: a base, a gold thin film on the base, and a decorative layer formed on the gold thin film. The gold thin film is formed through sputtering deposition, and has a thickness of 200 to 400 nm; the decorative layer comprises Prussian blue and has a thickness of 35 to 110 nm, and at least a part of the Prussian blue exists in the form of spherical and/or cubic particles.

Description

Electrochemical biosensor electrode, sensor and preparation method thereof

Technical field

The invention belongs to the field of biological materials and electrochemical biosensors, and relates to a method for detecting

Preparation and detection of a sensor for hydrogen peroxide (H ₂ O ₂ ).

Background technique

Hydrogen peroxide (H ₂ O ₂ ) has a strong oxidation effect and is a reaction product of many biological oxidases, and is often present as a by-product of the enzyme oxidation reaction in living organisms. Quantitative measurement of H ₂ O ₂ can be used as an effective quantitative characterization method for many hormones and metabolites in living organisms, such as blood glucose, lactic acid, cholesterol, alcohol, and the like. Therefore, effective quantitative detection techniques for hydrogen peroxide have a wide range of applications in the fields of food processing, textile industry, paper bleaching, pharmaceuticals, clinical medicine, and disinfectant manufacturing.

At present, the analysis methods for H ₂ O ₂ mainly include fluorescence spectroscopy, chemiluminescence, spectrophotometry, and electrochemical detection. Among them, the electrochemical detection method has the advantages of simple operation, high detection sensitivity, strong detection selectivity and high cost-effectiveness. The electrochemical method for the detection of H ₂ O ₂ is based on a redox reaction between catalase and hydrogen peroxide. The reaction between the enzyme and hydrogen peroxide allows electron transfer, which in turn produces a current signal that is detectable and corresponds to the concentration of the substance to be detected. Horseradish peroxidase (HRP) and Prussian blue (PB) are widely used catalase enzymes at this stage. PB is an artificial catalase because it has a low operating voltage range and can alleviate the interference signal of other oxidative hormones and metabolites in the living body to detect hydrogen peroxide. At the same time, PB can also be prepared by electrochemical, screen printing and other methods, with extremely high cost-effectiveness.

The biosensor prepared by the electrochemical method is an electrochemical sensor that uses a current or a voltage intensity as a signal and an electrode as a signal conversion device to apply a certain potential or voltage or current for measurement. Electrochemical biosensors typically implement electrical energy input and electrical signal output in a three-electrode system, including a reference electrode, a working electrode, and a counter electrode (auxiliary electrode). The reference electrode is generally silver/silver chloride or calomel electrode (SCE), the working electrode is a conductive metal electrode, and the counter electrode is a platinum wire or a platinum plate electrode. There is a current between the working electrode and the counter electrode, and the voltage input and output of the control system between the working electrode and the reference electrode. The electrochemical method can perform electrodeposition of the PB functional layer and current response detection of hydrogen peroxide. In the electrodeposition process of the PB functional layer, the working electrode is an electrode having conductivity, such as platinum, gold, silver, and a glassy carbon electrode; and the hydrogen peroxide current response process uses an electrode having a PB functional layer on the surface as a working electrode. .

At present, the working electrode for the hydrogen peroxide electrochemical sensor is generally a glassy carbon electrode, a carbon paste, a gold piece or a platinum plate. During the application process, the process of encapsulating and connecting the wires must be performed, resulting in an excessively large electrode volume and difficulty in flexibility. Manufacturing and other issues. With the development of physical vapor deposition technology, magnetron sputtering has developed into an efficient method for producing metal thin films. The metal film prepared by magnetron sputtering has an adjustable thickness of 100-1000 nm and is widely used in the field of flexible device preparation. Therefore, the application of the metal film for the preparation of the biosensor has important application value for the flexible design of the sensor and thus the application comfort of the biosensor.

Citation 1, which discloses a method for preparing a hydrogen peroxide electrochemical sensor, which uses a surface of a glassy carbon electrode sheet to coat a multilayer modified film, the first layer of which is naphthol green. The detection limit of the sensor is 0.9 μM, and the corresponding linear range is 8 × 10 ^{-6 -} 1.8 × 10 ^-4 M.

Citation 2 discloses an electrochemical sensor which electrodeposits gold nanoparticles and magnetic ferroferric oxide on the surface of a glassy carbon electrode and discloses a detection limit of 50 nM.

Citation 3 discloses a hydrogen peroxide non-enzyme electrochemical sensor and a preparation method thereof, which are modified with noble metal nanoparticles such as gold. It uses ion beam deposition to deposit precious metals, and controls the deposition amount within 5 layers. The linear range of detection of hydrogen peroxide is 4-44M.

Citation 4 discloses a gold microsphere-titanium nitride nanotube array composite material and a preparation method thereof, and also provides an application of the composite material in preparing an enzyme-free hydrogen peroxide electrochemical sensor. It arranges titanium nitride nano-array tubes on a titanium nitride substrate and forms gold microspheres on the top of the array tube.

Citation 5, which uses cyclic voltammetry to electrochemically deposit a Prussian blue film on a platinum electrode in two different electrolytes, and measures the cyclic voltammetric behavior of the modified film in a potassium chloride solution. Electrochemical impedance spectroscopy of the seed film. The electrochemical impedance spectroscopy measurements of the modified Prussian blue film platinum electrode show that the deposition conditions and the thickness of the deposited film have an effect on the electron transport process.

Therefore, it can be seen that there is still room for improvement in the prior art for obtaining high detection sensitivity, wide detection linear range, low detection limit, good use durability, and a relatively convenient preparation method.

List of cited documents

Patent literature

Citation 1: CN102043002A

Citation 2: CN101986147A

Citation 3: CN103792271A

Citation 4: CN103952763A

Non-patent literature

Citation 5: "Electrodeposition of Prussian Blue Film and Its Electrochemical Impedance Spectroscopy", Zhang Fenfen, et al., Chemical Research and Application, Vol. 15, No. 2, April 2003.

Summary of the invention

Problems to be solved by the invention

In view of the above problems, the present invention has been made in an effort to provide an electrochemical biosensor electrode, and an electrochemical biosensor based on the electrode, and in particular, when used for detecting a hydrogen peroxide concentration, the detection sensitivity can be obtained, the linear range can be detected, And excellent in both detection limit and durability.

In addition, the present invention also provides a simple and effective preparation method for preparing the above electrochemical biosensor electrode, and an electrochemical biosensor based on the electrode.

Solution to solve the problem

The invention firstly provides an electrochemical biosensor electrode, the electrode comprising: a substrate, a gold film on the substrate, and a finishing layer formed on the gold film,

The gold thin film is formed by a sputtering deposition method and has a thickness of 200 to 400 nm.

The modified layer includes Prussian blue and has a thickness of 35-110 nm.

At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.

According to the sensor electrode described above, the substrate is selected from the group consisting of graphite, carbon nanotubes, graphene, diamond-like carbon, a carbon substrate such as boron doped diamond, a glassy carbon substrate or a silicon substrate, ITO (indium tin oxide), A semiconductor transparent conductive film such as IZO (indium tin oxide), AZO (aluminum-doped zinc oxide), FTO (fluorine-doped tin oxide), or a conductive polymer film is preferably a silicon substrate.

According to the sensor electrode described above, a metal transition layer is present between the substrate and the gold thin film, and the metal transition layer contains at least one selected from the group consisting of Cr, Ti, and alloys thereof.

According to the sensor electrode described above, the metal transition layer is formed by a sputtering deposition method and has a thickness of 10 to 40 nm.

According to the sensor electrode described above, the modified layer is formed by an electrochemical deposition method.

In another aspect, the invention provides an electrochemical biosensor based on a sensor electrode according to any of the above.

In another aspect, the present invention provides an electrochemical biosensor for detecting hydrogen peroxide, wherein the sensor is based on the sensor described above, and the sensitivity of the sensor for detecting hydrogen peroxide is 250-350 mA/Mcm ² , and the detection limit is Above 0.77 μM, the linear range is 1-X μM, and the X is a value greater than 1500 and below 4500. .

In addition, the present invention provides a method of preparing an electrochemical biosensor electrode, the method comprising:

a step of forming a substrate electrode, and a step of depositing a modifying layer on the substrate electrode,

The step of forming a substrate electrode includes the step of depositing a gold thin film on the substrate by a sputtering method,

The step of forming a modified layer on the substrate electrode is to form a modified layer by an electrochemical deposition method, the modified layer including Prussian blue,

The thickness of the gold film is 200-400 nm, and the thickness of the modified layer is 35-110 nm.

According to the above method, in the step of forming a substrate electrode, comprising the step of pre-depositing a metal transition layer comprising a metal selected from the group consisting of Cr, Ti, and alloys thereof before depositing a gold thin film on the substrate At least either.

According to the method described above, the metal transition layer is deposited by a sputtering method to a thickness of 10 to 40 nm.

Further, the present invention provides a method for preparing an electrochemical biosensor for detecting hydrogen peroxide, comprising the method according to any one of the above, wherein the detection sensitivity of the sensor is 250-350 mA/Mcm ² , and the detection limit The ratio is 0.77 μM or more, the linear range is 1-X μM, and the X is a value greater than 1500 and below 4500.

Effect of the invention

The invention deposits a film including Prussian blue on a magnetron sputtering gold film substrate by using an electrochemical deposition method, and obtains a wide linear detection range based on the Prussian blue film modification, high sensitivity, low detection limit, and excellent durability. And a highly selective electrochemical biosensor, especially suitable for the detection of hydrogen peroxide.

In addition, the electrochemical biosensor electrode provided by the invention and the preparation method of the electrochemical biosensor for detecting hydrogen peroxide have simple and effective processes, are more suitable for industrial mass production, and reduce manufacturing costs.

DRAWINGS

Figure 1: Surface structure diagram of a 30 nm thick hydrogen peroxide sensor prepared in Example 1. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 2 is a view showing the surface structure of a hydrogen peroxide sensor having a thickness of 50 nm prepared in Example 2. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 3 is a view showing the surface structure of a 80 nm thick hydrogen peroxide sensor prepared in Example 3. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Fig. 4 is a view showing the surface structure of a 106 nm hydrogen peroxide sensor prepared in Example 4. Figure (a) is a scanning electron microscope (SEM) image, Figure (b) is a PB film and gold film interface measured by atomic force microscopy, and Figure (c) is the height information of the line segment shown in Figure (b).

Figure 5: A hydrogen peroxide sensor prepared in Examples 1-4 detects a new energy map of hydrogen peroxide. Where a, b, c, and d are the hydrogen peroxide chronograph current curves of the hydrogen peroxide sensor described in Examples 1, 2, 3, and 4 under a constant pressure of -0.1 V (vs. Ag/AgCl).

Figure 6 is a graph showing the relationship between the current density and the hydrogen peroxide concentration of the hydrogen peroxide sensor prepared in Examples 1-4. Where a, b, c, and d are the relationship between the current density and the hydrogen peroxide concentration of the hydrogen peroxide sensor prepared in Examples 1-4, respectively, which are calculated and fitted by FIG.

Figure 7: Hydrogen peroxide sensors prepared in Examples 1-4 were tested for durability by cyclic voltammetry. Figures a-d are CV curve images taken at intervals of 100 cycles after 1000 cycles of voltammetry of the hydrogen peroxide sensor prepared in Examples 1-4, respectively.

Figure 8: Durability curve of the hydrogen peroxide sensor prepared in Example 1-4 calculated by Figure 7, the points in the curve are divided by the first cycle volt-ampere curve by the area of the cyclic voltammogram per 100 intervals The percentage of area obtained is calculated from Figure 7.

detailed description

The detailed description of the various embodiments of the present invention is set forth in the claims and claims For example, in the present invention, "M" is used to mean 1 mol/liter or 1 mol/L for the sake of brevity.

A first embodiment of the present invention provides an electrochemical biosensor electrode, the electrode comprising: a substrate, a gold film on the substrate, and a modifying layer formed on the gold film, the gold film being It was formed using a sputter deposition method.

Base

The substrate of the present invention is not particularly limited as long as it can achieve the above effects of the present invention. As the substrate conventional in the art can be used as long as it has the required conductivity and support.

The substrate of the present invention may be selected from the group consisting of carbon, glassy carbon substrates, and semiconductor-based substrates or conductive polymer-based substrates.

Examples of the carbon-based substrate include carbon substrates such as graphite, carbon nanotubes, graphene, diamond-like carbon, and boron-doped diamond. For the semiconductor, for example, a semiconductor transparent conductive film such as silicon or ITO, IZO, AZO, or FTO may be mentioned. Further, the conductive polymer film is obtained by processing a film or a sheet material formed of a conductive polymer. The conductive polymer is a type of polymer material in which a polymer having a conjugated π-bond is chemically or electrochemically "doped" to convert it from an insulator to a conductor. Polymer conductive materials are generally divided into two types: composite type and structural type: 1 composite type polymer conductive material, which is made of general-purpose polymer materials and various conductive materials by filling compounding, surface compounding or lamination. 2; structural polymer conductive materials. It refers to the polymer structure itself or a polymer material that has a conductive function after being doped. According to the conductivity, it can be divided into polymer semiconductors, polymer metals, and the like.

Further, the substrate of the present invention is preferably a silicon wafer from the viewpoint of obtaining an electrode having a high detection range, high sensitivity, and low detection limit.

In addition, the substrate can be subjected to the necessary cleaning and/or activation prior to use of the substrate of the present invention. The specific washing and activating means are not particularly limited, and can be carried out by a method common to the art as long as the final effect of the present invention is not impaired.

Gold film and metal transition layer

A method of sputter deposition in the present invention deposits a gold thin film on the above substrate.

Specifically, a method of magnetron sputtering deposition is used in the present invention. Sputter deposition is a technique in which a surface of a material is bombarded with a charge ion in a vacuum environment to deposit bombarded particles on the surface of the substrate. Magnetron sputtering deposition methods are generally classified into balanced magnetron sputtering and unbalanced magnetron sputtering. For the present invention, balanced magnetron sputtering is preferably used from the viewpoint of substrate size and process simplicity.

Balanced magnetron sputtering is commonly referred to as conventional magnetron sputtering. The effective control of the secondary electrons by the magnetic field, so that the disadvantage of the two-pole sputtering is its own advantages. The principle of balanced magnetron sputtering is to make the secondary electrons in the electromagnetic field perpendicular to each other, and are bound to the surface of the target to make a circular motion along the magnetic field line of the "racetrack", which improves the ionization rate of the gas even if the working pressure Decreasing to the order of 10 ^-1 -10 ^-2 Pa can still increase the plasma density, which can increase the incident ion density, which is beneficial to reduce the sputtering voltage and increase the deposition rate. The secondary electrons can only be separated after the energy is exhausted. The surface of the target falls on the anode, so the substrate avoids the bombardment of secondary electrons, and the temperature rise of the substrate is low and there is no damage. Balanced magnetron sputtering can be effectively applied to the surface modification of temperature-critical substrate materials.

In the present invention, the deposition thickness of the gold thin film is controlled to be 200 to 400 nm, preferably 200 to 350 nm, and more preferably 250 to 330 nm. If the thickness of the gold film is too small, there is a fear that the surface defects cannot be eliminated, and the detection performance of the sensor electrode is noisy or reproducible. However, if the thickness is too large, the performance of the sensor electrode cannot be significantly improved on the one hand, and the manufacturing cost is increased by the other invention.

Further, in a preferred embodiment of the present invention, a metal transition layer is previously deposited on the substrate prior to performing the above-described sputter deposition of the gold film.

Depending on factors such as the conditions of sputter deposition control, when the gold film is directly deposited on the substrate, there is a lattice mismatch in the gold/substrate interface, which in some cases will lead to the initial formation of the gold film. The length of time also causes surface defects and unevenness to form easily when depositing a gold film. In other cases, it may also result in the adhesion of the gold film deposited on the substrate to the substrate being affected, thereby causing undesirable sensor aging in subsequent electrochemical cycles.

Therefore, pre-forming a metal transition layer between the gold film and the substrate is advantageous in avoiding the above problems. That is, in a preferred embodiment of the present invention, the metal transition layer can well solve the lattice mismatch existing between the substrate and the gold film and increase the bonding of the gold film to the substrate.

The metal transition layer can be deposited by magnetron sputtering deposition as described above, and deposited to a thickness of 10-40 nm, preferably 20-30 nm. For the thickness of the metal transition layer, if it is too small, it will not function as a lattice match, and if it is too large, the economy will be deteriorated.

The material of the metal transition layer is not particularly limited as long as it can satisfy the improvement of the interface bonding property of the base/gold film. For example, it may be selected from at least any of Cr, Ti, and alloys thereof. Preferably, in the present invention, the material of the metal transition layer is different from or not containing gold, and particularly preferably, in the present invention, the metal transition layer is formed using Cr.

Finishing layer

The modifying layer of the present invention is an electrochemically active material layer having a specific selectivity. Among the modified layers of the present invention, Prussian Blue (PB) is included.

In some embodiments of the invention, a layer of Prussian blue is deposited electrochemically on top of the gold film. Typically, when depositing, a gold film can be used as the working electrode, Ag/AgCl as the reference electrode, Pt wire as the counter electrode, and Fe ³⁺ and Fe(CN) ₆ ⁴⁺ , K ⁺ and HCl. The mixed solution was electrochemically deposited as an electrolytic solution, and electrochemically deposited at a constant deposition voltage of 0.4 V (relative to Ag/AgCl electrode) to obtain a PB modified electrode.

By controlling the deposition time, the deposition thickness of the PB layer can be controlled. The thickness of the PB layer in the present invention is 35 to 110 nm. The deposition time may be from 15 to 240 s. In some cases, the deposition time may be from 10 to 260 s, as long as the desired deposition thickness as described above can be obtained, and the deposition time can be appropriately adjusted depending on the deposition conditions.

In the present embodiment, by controlling and adjusting the thickness of the PB deposition, it is possible to obtain an electrochemical hydrogen peroxide biosensor electrode having a high detection linear range, high sensitivity, low detection limit, good durability, and strong selectivity.

It has been proposed in some existing studies to reduce the deposition thickness of the PB layer to improve the sensitivity of the detection. However, the present inventors have found that the deposition thickness of the PB layer is too low, which may result in loss of detection sensitivity. The reason is presumed that due to the low thickness of the PB deposit layer, the PB deposit layer may not be uniformly covered, and some defects in the deposit layer during the PB deposition process may not be effectively repaired. Therefore, according to the viewpoint of the present invention, the thickness of the PB deposited layer should be 35 nm or more, preferably 40 nm or more, such thickness, which is advantageous in eliminating the phenomenon of uneven deposition of PB and at the same time enabling surface defects of the PB layer to be eliminated. When the thickness of the PB layer is less than 35 nm, especially when the thickness thereof is 30 nm or less, the loss of sensitivity is exhibited. Therefore, although the smaller the thickness of the PB layer is theoretically, the more favorable the interaction between PB and the film is, which is beneficial to improve the detection sensitivity. However, due to the influence of actual physical and chemical conditions, the effective thickness of PB has a certain threshold.

As the thickness of the PB deposit increases, the linear range of detection becomes significantly wider, but the sensitivity of the detection at this time shows a downward trend. The reason may be speculated that as the thickness of PB immersion increases, the defects on the surface of PB are improved. Therefore, a wide linear range can be exhibited at a constant voltage, and the linear correlation coefficient is greater than 0.99, but at this time PB The distance between the surface layer and the gold film becomes large, so that the distance between the detected molecule or its product interacting with PB diffuses into the gold thin film layer becomes longer (that is, the diffusion or receiving distance of the electric signal increases), and thus, the detection sensitivity is caused. The relative decline.

In addition, with the increase of the deposition thickness of PB layer, the microscopic morphology of PB transitions from mainly (amorphous) spherical to mainly cubic (crystalline) granular, and with the change of the morphology of PB layer, it affects Durability of the use of the electrode. In some cases, since the amorphous PB layer is mainly composed of amorphous spherical particles, the durability increases as the thickness of the PB increases, but the PB thickness increases to a certain extent and is within a certain PB thickness range. The durability no longer continues to increase significantly. However, after further deposition of the PB layer, cubic PB particles are gradually formed in the PB layer, which in turn leads to an increase in durability. When the cubic PB particles in the PB layer grow to a certain extent, cracks may occur on the surface of the PB layer due to the action of grain boundaries.

Therefore, it can be seen that for the deposition thickness of the PB layer on the gold thin film, it is necessary to consider not only excellent sensitivity but also a wide detection linear range, durability of use, and the like. Therefore, from this point of view, the PB deposition thickness of the present invention is 35-110 nm, preferably 40-70 nm, more preferably 40-60 nm.

By controlling the thickness of the above PB film, for example, the following excellent detection effect can be achieved, and the detection limit for hydrogen peroxide is 0.77 μM or more, that is, the minimum value of the detection limit is 0.77 μM; the linear range of detection is 1-X μM, The X is a value greater than 1500 and below 4500, and, in some embodiments, the linear range is preferably 1-3000 μM, the detection sensitivity is 250-350 mA/Mcm ² , preferably 280-350 mA/Mcm ² , more preferably It is 310-350 mA/Mcm ² .

Other active substances

In the present embodiment, in addition to the electrochemical deposition of PB on the gold film as the active material layer, without limitation, other activities may be deposited in the same layer or in a separate layer other than the PB layer according to actual needs. substance.

The active substance may be various enzymes. There may be mentioned one or more of glucose oxidase, lactate oxidase, horseradish catalase, cholesterol oxidase, xanthine oxidase, acetylcholinesterase, and organophosphine hydrolase.

The above active materials can be deposited by conventional deposition methods in the art. Further, one or more layers of deposition may be performed as needed, provided that the technical effects of the present invention are not impaired.

In a second embodiment of the invention, an electrochemical biosensor is provided. The sensor is based on a sensor electrode as described or defined in <First Embodiment>.

The above sensors can be used directly as direct sensors to detect small molecules sensitive to PB, such as hydrogen peroxide.

Further, as described above, a sensor including another active material layer can be used as an indirect sensor. The molecules or components such as glucose, protein, and the like can be detected by using the enzymes mentioned above in combination.

Further, the above-described sensor may be subjected to necessary encapsulation or any other modification as long as the effects of the present invention are not impaired. Through the necessary packaging, it can be applied to various occasions, and it is beneficial to improve the safety of use and ensure the effectiveness of detection.

Further, in the embodiment of the present invention, an electrochemical biosensor including the sensor electrode in <First Embodiment> is provided at the same time, and based on this, is prepared as a working electrode. In addition, the sensor further includes a reference electrode and a counter electrode. The reference electrode can be a conventional reference electrode in the art, such as a calomel electrode, a silver/silver chloride electrode, and the like. For the counter electrode, a Pt wire or a plate or the like can be used.

Typically, the product of the present embodiment is an electrochemical biosensor that can directly detect the concentration of hydrogen peroxide. Specifically, in the same electrolytic cell, the present embodiment adopts a three-electrode system, a working electrode, a reference electrode, and a counter electrode, wherein the working electrode is a magnetron sputtering gold film. Thereafter, a certain amount of deposition liquid is added to the electrolytic cell, and a constant voltage is applied to the working electrode, and a Prussian blue film is deposited on the surface of the gold film after a certain period of time. The obtained Prussian blue modified electrode was subjected to a certain chemical and physical process, and then placed in the same electrolytic cell, and the Prussian blue modified electrode was used as a working electrode to measure the hydrogen peroxide concentration. During the measurement, a constant voltage is still applied to the working electrode, a certain amount of phosphate buffer solution (PBS) is added to the electrolytic cell, and different concentrations of hydrogen peroxide solution are dropped under stirring, and the current intensity response is detected.

In this embodiment, the present invention provides a method of preparing an electrochemical biosensor electrode and preparing an electrochemical biosensor for detecting hydrogen peroxide.

Wherein, the method for preparing the sensor electrode comprises:

According to the above method, in the step of forming the substrate electrode, further comprising the step of pre-depositing a metal transition layer comprising Cr, Ti, and an alloy thereof before depositing the gold thin film on the substrate At least either.

In addition, the method for preparing an electrochemical biosensor for detecting hydrogen peroxide according to the present embodiment includes the above steps, and optionally, may further include a step of forming or preparing another active material layer, preparing a reference electrode, a counter electrode, and optionally The necessary packaging steps.

In these steps, the apparatus or equipment to be used is not particularly limited as long as it satisfies the effects sufficient to achieve the present invention.

More specifically, the technical solution for preparing the electrochemical biosensor electrode in the present embodiment is as follows:

(1) Formation of the base electrode: a silicon wafer having a thickness of 300 μm is placed in a magnetron sputtering apparatus, and ultra-pure chromium (chromium content ≥99.99 wt.%) is used as a target, and a layer of chrome metal having a thickness of 20 nm is first deposited. Then, a gold film with a thickness of 200-400 nm was sputter deposited with gold (gold content ≥99.99 wt.%) as a target.

(2) The obtained gold film was formed into a square gold film electrode having a size of 10 mm × 10 mm by a cutting process, and ultrasonically washed and blown dry in dilute hydrochloric acid, dilute sodium hydroxide solution, deionized water, acetone and alcohol.

(3) Using the pretreated gold film as the working electrode, Ag/AgCl as the reference electrode, Pt wire as the counter electrode, and the mixture containing Fe ³⁺ and Fe(CN) ₆ ⁴⁺ , K ⁺ and HCl The solution was electrochemically deposited for the electrolyte, the deposition time was 10-240 s, and the deposition voltage was 0.4 V (relative to the Ag/AgCl electrode) to obtain a PB modified electrode.

(4) The obtained PB modified electrode is subjected to cyclic voltammetry activation in a solution containing K ⁺ and HCl, the voltage range is -0.05 V to 0.35 V, the voltage scanning rate is 0.05 V/s, and the number of cycles is 25-40, preferably 35 times. Then, the potentiostatic polarization is carried out in a phosphate buffer, the constant potential is -0.1 to 0.1 V (relative to the Ag/Cl electrode), preferably -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time is 120-600 s, preferably 600s. The PB modified electrode was then washed with deionized water and dried at 100 ° C for 1 h to obtain the electrochemical biosensor electrode.

Further, the deposition solution in the step (3) is 2.5 mM FeCl ₃ + 2.5 mM K ₃ Fe(CN) ₆ + 0.1 M KCl + 0.12 M HCl solution, and the above inorganic salt and acid solution are prepared with 100 mL of deionized water. .

Further, the deposition time in the step (3) is 10 s, the deposition voltage is 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film is 30 nm.

Further, the deposition time in the step (3) was 40 s, the deposition voltage was 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film was 50 nm.

Further, the deposition time in the step (3) is 120 s, the deposition voltage is 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film is 80 nm.

Further, the deposition time in the step (3) was 240 s, the deposition voltage was 0.4 V (relative to the Ag/AgCl electrode), and the thickness of the PB film was 106 nm.

Further, the electrode stabilizing solution in the step (4) is 0.12 M HCl + 0.1 M KCl solution, which is prepared from the above inorganic salt and concentrated hydrochloric acid solution and 100 mL of deionized water.

Further, the phosphate buffer in the step (4) is a 0.05 M KH ₂ PO ₄ /K ₂ HPO ₄ +0.1 M KCl solution, which is prepared from the above inorganic salt powder and 100 mL of deionized water.

In the present embodiment, the above electrode can be used as a direct sensor for the detection of hydrogen peroxide. In another embodiment, with other active substance enzymes, as described above, detection of substances such as glucose, protein, etc. (ie, indirect) can be achieved. sensor).

The functional film (PB film) of the hydrogen peroxide sensor electrode of the present invention has a thickness of 35-110 nm, preferably 40-70 nm, more preferably 40-60 nm, and the detection limit for hydrogen peroxide is 0.77 μM or more, and the linear range of detection is 1 -XμM, said X being a value greater than 1500 and below 4500, in some embodiments the linear range is preferably 1-3000 μM, the detection sensitivity is 250-350 mA/Mcm ² , preferably 280-350 mA/Mcm ² , more preferably It is 310-350 mA/Mcm ² .

Compared with the prior art, the beneficial effects of the present invention are mainly embodied in: the present invention adopts an electrochemical deposition method to prepare a hydrogen peroxide sensor based on a PB functional layer, which has high sensitivity, wide linear range, low detection limit and excellent durability. The preparation and fixing of the functional layer is completed in one time. Compared with other sensors, the invention adopts a magnetron sputtering gold film, which has the advantages of low cost, simple operation, and comfortable living environment for flexible preparation and application.

Example

The invention is further described below in conjunction with specific embodiments, but the scope of protection of the invention is not limited thereto.

The sensitivity and durability test method for detecting hydrogen peroxide by the hydrogen peroxide sensor of the present invention is as follows:

(1) Sensitivity and detection range test:

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. In the 100 mL phosphate buffer (pH=6.2) described in the step (3), under a slight stirring condition (rotor rotation speed of 200-250 RMP), a chronograph current method is used, and a quantitative concentration of hydrogen peroxide solution is continuously added for sensitivity. And linear range test, a constant potential is applied to the working electrode during the experiment, and the voltage range is -0.15 to 0.15 V (relative to the Ag/Cl electrode), preferably -0.1 V.

(2) Durability test:

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. The durability of the sensor was tested by cyclic voltammetry in 100 mL of phosphate buffer (pH = 6.2) as described in step (3). The cyclic voltammetry voltage range is -0.05 to 0.35 V (relative to the Ag/AgCl electrode), the scanning rate is 50 mV/s, and the number of cycles is 600-1200 times, preferably 1000 times, by calculating the cyclic voltammogram area of each 100 times. Durability test.

Example 1 (Reference example)

(1) Magnetron sputtering gold film treatment

A silicon wafer with a thickness of 200-400 μm is placed in a magnetron sputtering apparatus, and ultra-pure chromium (chromium content ≥99.99 wt.%) is used as a target. First, a layer of chrome metal having a thickness of 20 nm is deposited; then gold (gold) A content of ≥99.99 wt.% is used as a target, and a gold film having a thickness of 200-400 nm is sputter deposited. After that, the obtained magnetron sputtered gold film was cut into a 10 mm×10 mm square gold film electrode, and ultrasonically cleaned in dilute hydrochloric acid, dilute sodium hydroxide solution, deionized water, acetone and alcohol for 30 min, and then a nitrogen spray gun was used. Blow dry and obtain the pretreated gold film for use.

(2) Preparation of Prussian blue modified film

Using a three-electrode system, the gold film base material pretreated by step (2) is used as a working electrode, Ag/AgCl is used as a reference electrode, Pt wire is used as a counter electrode, and a final concentration of 2.5 mM FeCl _{3 is used} . 2.5 mM K ₃ Fe(CN) ₆ , a final concentration of 0.1 M KCl and a final concentration of 0.12 M HCl solution for electrochemical deposition of the electrolyte, deposition time of 10 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) A PB modified electrode having a thickness of 30 nm was obtained.

(3) The obtained PB modified electrode was subjected to cyclic voltammetry activation in a mixed solution of HCl having a final concentration of 0.12 M and a final concentration of 0.1 M in KCl, the voltage range was -0.05 to 0.35 V, and the voltage scanning rate was 0.05 V/s. The number of cycles is 25. Then, in a 100 mL phosphate buffer (pH=6.2) containing 0.05 M KH ₂ PO ₄ /K ₂ HPO ₄ +0.1 M KCl, constant potential stable polarization, constant potential -0.05 V (relative to Ag/Cl electrode), stable The polarization time is 120s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gun, and dried at 100 ° C for 1 h to obtain the hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG.

(4) Hydrogen peroxide current response test:

A hydrogen peroxide biosensor with a PB functional layer thickness of 30 nm was placed in 0.05 M phosphate buffer (pH=6.2), and under a slight agitation condition (rotor rotation speed of 200 RMP), a chronoamperometry method was used to continuously add a quantitative concentration. The hydrogen peroxide solution was tested for sensitivity and linear range, and a 0.1 V (relative to Ag/Cl electrode) constant potential was applied to the working electrode during the experiment. The results are shown in the curve a in Fig. 5 and the curve a in Fig. 6. The detection limit of the sensor for hydrogen peroxide is 0.44 μM (signal-to-noise ratio is 3), the linear range is 1-1500 μM, and the detection sensitivity is 204 mA/Mcm ^{2 .} .

(5) Durability test of hydrogen peroxide biosensor

The hydrogen peroxide sensor (Au/PB electrode) of the present invention is used as a working electrode, Ag/Cl is a reference electrode, and the Pt sheet is a counter electrode. The durability of the sensor was tested by cyclic voltammetry in phosphate buffer (pH = 6.2). Cyclic voltammetry has a voltage range of -0.05 to 0.35 V (relative to Ag/AgCl electrode), a scan rate of 0.05 V/s, and a number of cycles of 1000 cycles, every 100 cycles of the volt-ampere curve and the area of the first cyclic voltammetry curve. The ratio is calculated to obtain the cyclic voltammogram shown in a of Fig. 7 and the a curve in Fig. 8. Experimental data shows that the capacitor has a capacitance decay rate of about 35% after 1000 cycles of volt-ampere scanning.

Example 2

Using a three-electrode system, the gold film base material pretreated in the step (1) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 1 was used. Electrochemical deposition, deposition time of 40 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 50 nm.

The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 1, and the voltage range was -0.05 V to 0.35 V, the voltage scanning rate was 0.05 V/s, and the number of cycles was 35. Thereafter, potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 1, a constant potential of -0.05 V (relative to the Ag/Cl electrode), and a stable polarization time of 600 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gun, and dried at 100 ° C for 1 h to obtain the hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG.

The detection sensitivity and linear range of the sensor for hydrogen peroxide (detection method is the same as in the first embodiment) are shown in the curve b in Fig. 5 and the curve in b in Fig. 6. The detection limit of hydrogen peroxide is 0.77 μM (the signal-to-noise ratio is 3), the linear range is 1-3000 μM, and the detection sensitivity is 341 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 1) is shown in Figure 7 b and Figure 8 b curve, the capacitance decay rate after 1000 cycles of voltammetry scanning is less than 25%.

Example 3

Using a three-electrode system, the gold film base material pretreated in the step (1) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 1 was used. Electrochemical deposition, deposition time of 120 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 80 nm.

The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 1, and the voltage range was -0.05 V to 0.35 V, the voltage scanning rate was 0.05 V/s, and the number of cycles was 35. Thereafter, the potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 1, the constant potential was -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time was 240 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gun, and dried at 100 ° C for 1 h to obtain the hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG.

The sensitivity and linear range of the sensor for hydrogen peroxide (see Example 1 for the detection method) are shown in the c curve in Figure 5 and the c curve in Figure 6. The detection limit of hydrogen peroxide is 0.1 μM (the signal-to-noise ratio is 3), the linear range is 1-3500 μM, and the detection sensitivity is 318 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 1) is shown in Figure 7 c and the curve c in Figure 8, and the capacitance decay rate after 1000 cycles of voltammetric scanning is 25%.

Example 4

Using a three-electrode system, the gold film base material pretreated in the step (1) of Example 1 was used as a working electrode, Ag/AgCl was used as a reference electrode, and Pt wire was used as a counter electrode, and the electrolyte in Example 1 was used. Electrochemical deposition, deposition time of 240 s, deposition voltage of 0.4 V (relative to Ag / AgCl electrode) to obtain a PB modified electrode with a thickness of 106 nm.

The obtained PB modified electrode was subjected to cyclic voltammetry activation in the activation solution of Example 1, and the voltage range was -0.05 V to 0.35 V, the voltage scanning rate was 0.05 V/s, and the number of cycles was 30. Thereafter, the potentiostatic polarization was carried out in the phosphate buffer (pH = 6.2) in Example 1, the constant potential was -0.05 V (relative to the Ag/Cl electrode), and the stable polarization time was 240 s. Thereafter, the PB modified electrode was washed with deionized water, dried with a nitrogen gun, and dried at 100 ° C for 1 h to obtain the hydrogen peroxide biosensor. The surface structure and functional layer thickness of the hydrogen peroxide biosensor are shown in FIG.

The detection sensitivity and linear range of the sensor for hydrogen peroxide (see Example 1 for the detection method) are shown in the d curve of Fig. 5 and the d curve of Fig. 6, and the detection limit of hydrogen peroxide is 2.38 μM (the signal-to-noise ratio is 3), the linear range is 5-4500 μM, and the detection sensitivity is 281 mA/Mcm ² . The durability test of the electrode (test method is the same as in Example 1) is shown in Figure 7 d and the d curve in Figure 8, and the capacitance decay rate after 1000 cycles of voltammetric scanning is less than 22.5%.

Example 5 (Reference example)

The experimental conditions of Example 4 were repeated except that the time for depositing the PB film was adjusted to 280 s to obtain a thicker PB deposited film.

As can be seen from the above Examples 1-4, as the deposition time increases, the deposition thickness of the PB film gradually increases.

As for the determination that the thickness of the active material layer is smaller as indicated in the prior literature, the sensitivity is about high. Example 1 shows that the deposition thickness (30 nm) is too small at the deposition time of 10 s, possibly depositing on the surface of the gold film. The layer appears unevenly distributed or produces too many defects, resulting in low detection sensitivity. This also shows that although the above qualitative consensus may exist to some extent in the art, from the quantitative analysis of the present invention, the inverse correlation of the PB film thickness to the detection sensitivity has a certain threshold. That is, only when the thickness exceeds this threshold, the PB film thickness exhibits an inverse correlation with the detection sensitivity.

With respect to Example 2-4, it was revealed that as the thickness of the PB film was increased, although the detection sensitivity was sequentially lowered, the detection linear range was gradually widened, and therefore, the PB film can be obtained according to the data of Examples 2-4. The thickness is controlled in an appropriate range, and a good detection sensitivity can be obtained, and a wide detection range can be achieved.

For the detection limit, although Example 1 shows a lower detection limit, considering its low detection sensitivity and the possibility that its PB film may be defective, its detection sensitivity is not within the scope of the present invention.

For Example 5, the same Example 5 was repeated while actually operating, and it was found that cracks appeared on the surface of some PB films, resulting in dispersion and instability of the test data, which may be due to an increase in deposition thickness (especially After the PB film thickness is greater than 110 nm, the cubic crystals in the PB film layer continue to grow, and the separation between the grain boundaries and the grain boundaries is related.

Industrial availability

The bioelectrochemical sensor of the present invention can be industrially produced and can be applied to the detection of hydrogen peroxide in an organism.

Claims

An electrochemical biosensor electrode, characterized in that the electrode comprises: a substrate, a gold film on the substrate, and a finishing layer formed on the gold film,

The gold thin film is formed by a sputtering deposition method and has a thickness of 200 to 400 nm.

The modified layer includes Prussian blue and has a thickness of 35-110 nm.

At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.
The sensor electrode according to claim 1, wherein the substrate is selected from the group consisting of a carbon substrate, a glassy carbon substrate, a semiconductor substrate, or a conductive polymer film substrate.

The carbon substrate is selected from the group consisting of graphite, carbon nanotubes, graphene, diamond-like carbon or boron-doped diamond;

The semiconductor substrate is selected from the group consisting of: a silicon substrate, or an ITO, IZO, AZO, FTO semiconductor transparent conductive film;

As a preferred substrate, it is a silicon substrate.
The sensor electrode according to claim 1 or 2, wherein a metal transition layer is present between the substrate and the gold thin film, the metal transition layer comprising at least one selected from the group consisting of Cr, Ti, and alloys thereof.
The sensor electrode according to claim 3, wherein the metal transition layer is formed by a sputtering deposition method and has a thickness of 10 to 40 nm.
The sensor electrode according to any one of claims 1 to 4, wherein the modified layer is formed by an electrochemical deposition method.
An electrochemical biosensor obtained by the sensor electrode according to any one of claims 1-5.
An electrochemical biosensor for detecting hydrogen peroxide, characterized in that the sensor is the sensor according to claim 6, the sensitivity of the sensor for detecting hydrogen peroxide is 250-350 mA/Mcm 2 , and the detection limit is 0.77 Above μM, the linear range is 1-X μM, and the X is a value greater than 1500 and below 4500.
A method for preparing an electrochemical biosensor electrode, characterized in that the method comprises:

a step of forming a substrate electrode, and a step of depositing a modifying layer on the substrate electrode,

The step of forming a substrate electrode includes the step of depositing a gold thin film on the substrate by a sputtering method,

The step of forming a modified layer on the substrate electrode is to form a modified layer by an electrochemical deposition method, the modified layer including Prussian blue,

The thickness of the gold film is 200-400 nm, and the thickness of the modified layer is 35-110 nm.

At least a portion of the Prussian blue is present in the form of spherical and/or cubic particles.
The method according to claim 8, wherein in the step of forming the substrate electrode, further comprising the step of pre-depositing a metal transition layer before the deposition of the gold film on the substrate, the metal transition layer comprising a layer selected from Cr At least any of Ti, Ti, and alloys thereof.
The method according to claim 9, wherein the metal transition layer is deposited by a sputtering method to a thickness of 10 to 40 nm.
A method for preparing an electrochemical biosensor for detecting hydrogen peroxide, comprising the method according to any one of claims 8 to 10, wherein the detection sensitivity of the sensor is 250-350 mA/Mcm 2 , and the detection The limit is 0.77 μM or more, the linear range is 1-X μM, and the X is a value greater than 1500 and below 4500.